European Journal of Agronomy 12 (2000) 281 – 289
www.elsevier.com/locate/euragr
Root-released organic acids and phosphorus uptake of two
barley cultivars in laboratory and field experiments
Tara S. Gahoonia a,*, Farouq Asmar b, Henriette Giese b,
Gunnar Gissel-Nielsen b, Niels Erik Nielsen a
a
The Royal Veterinary and Agricultural Uni6ersity, Department of Agricultural Sciences,
Plant Nutrition and Soil Fertility Laboratory, Thor6aldsens6ej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark
b
Risø National Laboratory, Department of Plant Biology and Biogeochemistry, PO Box 49, DK-4000 Roskilde, Denmark
Received 6 September 1999; received in revised form 18 January 2000; accepted 11 February 2000
Abstract
A major portion of phosphorus (P) applied as fertilizers is bound in soils as P compounds of variable adsorption
strength, reducing the effectiveness of P fertilization. Plant genotypes equipped with mechanisms for utilizing the
adsorbed P more efficiently can, therefore, enhance the effectiveness of P fertilization. Such genotypes will also enrich
plant gene pools for further analysis and upgrading of P efficiency by selection and breeding. We studied the variation
and the mechanisms of P uptake of two winter barley (Hordeum 6ulgare L.) cultivars Marinka and Sonate (parents
of existing 200 haploid progeny lines), by laboratory and field experiments. After cultivation in nutrient solution for
21 days, Marinka produced more roots than Sonate, but similar amounts of dry shoots of lower P content (Marinka
3.4 90.4 mg g − 1, Sonate 4.9 90.6 mg g − 1). The total P uptake per plant did not differ between the cultivars.
Marinka retained more P in roots as indicated by the higher concentration of P in the roots (Marinka 3.9 90.3 mg
g − 1 and Sonate 3.0 90.4 mg g − 1). In sterile nutrient solution culture, the cultivars differed mainly in release of
organic acids from the roots, with Marinka releasing three times more citric acid and nearly two times more acetic
acid than Sonate. The cultivars had similar root hair lengths and they did not differ (P\ 0.05) in depletion of
available soil P fraction (extracted with 0.5 M NaHCO3) in the rhizosphere. Marinka absorbed nearly twice as much
P from the strongly adsorbed soil P fraction (extracted with 0.1M NaOH). Also under field conditions, Marinka
absorbed more P and produced more shoot dry matter. The higher P uptake by Marinka than Sonate can be
attributed to its ability to acquire P from strongly adsorbed soil P by releasing more organic acids, especially citric
acid, from its roots. © 2000 Elsevier Science B.V. All rights reserved.
Keywords: Barley genotypes; Organic acids; Phosphorus efficiency; Rhizosphere; Root exudation; Root hairs
1. Introduction
* Corresponding author. Tel.: +45-35283497, fax: +4535283460.
E-mail address: [email protected] (T.S. Gahoonia)
Concerns about depletion of high-grade phosphate rock, the necessary fertilizer raw material
1161-0301/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.
PII: S 1 1 6 1 - 0 3 0 1 ( 0 0 ) 0 0 0 5 2 - 6
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T.S. Gahoonia et al. / Europ. J. Agronomy 12 (2000) 281–289
(Cathcart, 1980), cost of P fertilization in developing countries and potential environmental problems associated with P inputs in developed
countries, have stimulated the search for means of
saving and utilizing P more efficiently. In addition
to the agronomic measures (Mengel, 1997), the
need of selecting and breeding P-efficient crop
varieties is receiving increased attention (Lynch,
1998). Soil phosphorus problems are particularly
severe in developed countries, where rapid growth
in human population is expected. For securing
food to the increasing population, the need for P
fertilization is also expected to increase (Brynes
and Bumb, 1998). However, when P fertilizers are
applied to replenish soil fertility, about 70 – 90%
of the P fertilizers is adsorbed and retained in soil
in various P compounds. The mechanisms of P
adsorption in soils are not fully resolved (Sample
et al., 1980), but for addressing the bioavailability
of soil P, it can be divided into easily desorbable
plant available P and strongly adsorbed P fractions by using sequential extraction procedure
(Hedley et al., 1982). Although, in the long-term,
the adsorbed soil P may not be considered lost,
but as a phosphorus capital (Sanchez and Leakey,
1997), it is not immediately available for supporting plant growth. This reduces the effectiveness of
P fertilization, inducing the lack of economic incentives to apply costly P fertilizers (Brynes and
Bumb, 1998).
Plant species and their cultivars possess diverse
root morphological (Gahoonia et al., 1997) and
physiological (Neumann et al., 1999) processes for
adapting to low P supply, but the relative importance of the P mobilizing processes may differ
with plant species and cultivars. For example,
white lupine (Braum and Helmke, 1995) and Pigeon pea (Ae et al., 1990) possesses specific ability
to mobilize and use adsorbed soil P, not available
to other plants. Plant roots release a variety of
organic acids in the rhizosphere (Jones and Darrah, 1995). The release increases during P stress
(Lipton et al., 1987; Hoffland et al., 1992). Bolan
et al. (1990) reported that organic acids increase
soil P availability by decreasing adsorption of P
and by increasing dissolution of relatively insoluble P compounds. Other reports (Dinkelaker et
al., 1995) suggest that root-released citric acid
increase the availability of mineral-bound P by
solubilizing Ca, Fe, and Al phosphates.
The role of chemical organic acids (Gerke,
1992; Hue, 1991) and root-released organic acids
by different plant species in mobilization of soil P
is documented (Jones and Darrah, 1994). However, the information is sparse (Römer and
Schenk, 1998), whether the release of organic
acids from roots of barley cultivars differs and
how it affects rhizosphere P mobilization and
uptake by plants. Such information is important
for elucidating the plant genetic factors, which
may increase the release and uptake of P from
insoluble soil P fractions, improving the effectiveness of P fertilization. In this paper, we report the
variation in root-released organic acids and their
relationship to phosphorus uptake of two barley
cultivars Marinka and Sonate in laboratory and
field studies.
2. Materials and methods
2.1. Soil
The soil used for the field and rhizosphere
studies was a sandy clay loam, which had received
no P since 1966. The soil contained 15% clay, 18%
silt, 65% sand, total C= 11.5 mg g − 1, total N=
1.3 mg g − 1. Inorganic P extractable with 0.5 M
NaHCO3 (NaHCO3-Pi) was 0.45 mmole P kg-1
soil and inorganic P extractable with 0.1 M
NaOH (NaOH-Pi) was 2.45 mmole P kg − 1 soil.
The soil solution P concentration was 3 mM. Soil
pH (0.01 M CaCl2) was 5.6 and cation exchange
capacity=8.4 cmolc kg − 1 soil at pH 7.
2.2. Culti6ars
The two winter barley cultivars (Hordeum 6ulgare L. cv. Marinka and Sonate) were chosen for
the study, because 200 double haploid progeny
lines of crosses Marinka× Sonate and basic maps
including Restriction Fragment Length Polymorphism (RFLP) markers are available. Therefore, if
differences in P uptake between the two cultivars
are characterized, the progeny lines can be useful
for genetic analysis of P efficiency in barley.
T.S. Gahoonia et al. / Europ. J. Agronomy 12 (2000) 281–289
2.3. Nutrient solution experiments
2.3.1. Root length determination
Because of concerns of persistent holes in the
small low-P plots (also used for other purposes)
sampling of soil cores for root studies was not
done. The ability of the cultivars to produce roots
was determined in a nutrient solution experiment.
Plants were grown in a basic nutrient solution
(Table 1) in 5 l plastic pots in a glass-house as
described by Gahoonia and Nielsen (1997). The
electrical conductivity of the solution was maintained at 0.63 mS cm − 1 by adding a maintenance
solution (Table 1). Roots were harvested after 21
days and total root system length was measured
using a scanner (ScanJet IIcx) and Dt-Scan software (Delta-T Devices, Cambridge, England). All
shoots and roots were washed thoroughly with
distilled water for determining P concentration.
2.3.2. Determination of root-released organic
acids
To assess the variation in root-released organic
acids, 40 seeds of each cultivar were sown individually in sterile sand in Eppendorf vials and supplied with sterile nutrient solution as described by
Asmar and Gissel-Nielsen (1996). After 7 days,
the Eppendorf vials with the seedlings were placed
in the holes (10 mm diameter) made on the lids of
35 ml bottles, containing 30 ml of the sterile
maintenance nutrient solution (Table 1), where
the P concentration was reduced to 0.005 mM.
The bottles were wrapped in aluminum foils and
transferred to a growth chamber with a night/day
regime of 8/16 h, 15/20°C, relative humidity 65 –
75%, and light intensity 460 mE s − 1 m − 2. After
10 days, the roots of the plants were first rinsed
2 – 3 times with a sterile 225 mM CaCl2 solution
(pH 4.3) for 30 min in a laminar-flow-hood. The
bottle lids with the plants were transferred into
10-ml sterile bottles. The roots protruding from
the Eppendorf vials were dipped into 7 ml of a
sterile 450 mM CaCl2 solution (pH 7). The CaCl2
solution maintains the structural integrity of root
membranes during the growth under P stress.
After 12, 24 and 48 h, 12 plants of each cultivar
were harvested, and the solutions were filtered
through millipore filters, then freeze-dried and
283
extracted with 20 and 98% ethanol for 30 min at
80°C (Jones and Darrah, 1995). The solutions
were analyzed for organic acids with HPLC using
Aminex HPX-87H column. Filtrate samples (100
ml) were injected into the HPLC using a glass
syringe and eluted isocratically with 4 mM H2SO4
at a constant flow rate 0.6 ml/min for 20 min at
20°C. Peaks for the organic acids were detected at
a wavelength of 210 mm and the organic acids
were identified by comparison to the retention
times obtained for pure organic acids injected as
standards. From the peak areas, the quantity of
organic acids in the samples was calculated and
expressed as mg organic acid, g − 1 root dry
weight, day − 1.
2.4. Rhizosphere experiment
To study P acquisition from different soil P
fractions in the rhizosphere, soil samples of
known distances from roots were obtained by
thin-slicing the rhizosphere soil as described by
Gahoonia and Nielsen (1991). The cultivars were
first pre-grown for 12 days in vermiculite filled in
PVC tubes (length 10 cm, diameter 4.4 cm) closed
at the bottom by nylon cloth, which was impervious to roots. Two ceramic fiber wicks were placed
along the inner sides of the tubes to supply nutrient solution of a defined composition. The tubes,
along with the plants having uniformly developed
root mats were transferred to soil columns filled
into PVC tubes (length 3 cm, diameter 5.6 cm). A
nylon screen of mesh size 53-mm (open space,
22%) separated each column into 3-cm test soil
below and 1-cm soil layer above the screen. The
soil columns (bulk density 1.3 g cm − 3) were
maintained at defined moisture (u= 0.21) by placing them over small, cup-shaped sand baths, each
fitted with a wick dipping into a distilled water
reservoir. For some columns, six 1-mm holes were
made in the nylon screen to measure root hairs on
the roots grown in the soil as described in the next
section. After transplantation, new root mats developed over the nylon mesh, representing a root
surface area of 24.6 cm − 2. No roots, but only
root hairs penetrated into the soil. The supply of
the maintenance solution (Table 1) to the plant
roots in the vermiculite was continued via the two
284
Nutrients
NO3 (mM)
NH4 (mM)
P (mM)
S (mM)
K (mM)
Ca (mM)
Mg (mM)
Cl (mM)
Na (mM)
Fe (mM)
Mn (mM)
Zn (mM)
Cu (mM)
B (mM)
Mo (mM)
Basic
Maintenance
5.0
13
0.0
3.2
0.005
0.54
0.44
0.61
0.58
7.16
1.92
1.40
0.78
0.35
0.10
0.40
0.001
0.24
50
15
7.0
12
0.7
6.0
0.7
0.9
2.0
4.6
0.7
0.2
T.S. Gahoonia et al. / Europ. J. Agronomy 12 (2000) 281–289
Table 1
The composition and concentration of basic and maintenance solutions
T.S. Gahoonia et al. / Europ. J. Agronomy 12 (2000) 281–289
285
wicks at 20-cm water tension. The solution was
adjusted to match the average nutrient ratio in
dry matter of cereals. Based on previous studies
(Gahoonia and Nielsen, 1991, 1992), the solution
creates moderate P deficiency and avoids deficiencies of other nutrients. The equal water uptake by
plants from the external nutrient solution maintained equal supply of nutrients to both the cultivars. Water uptake also occurred via the soil
column (1092% of the total water uptake). The
percentage of total N as ammonium in the supply
solution was adjusted to maintain a constant rhizosphere soil pH (Gahoonia and Nielsen, 1992).
The experiments were conducted under controlled
conditions (light intensity 280 mE s − 1m − 2, light/
dark period 16/8 h, temperature 18/15°C, relative
humidity 75%). After 14 days, the soil columns
were separated from the root mats, quickly frozen
in liquid nitrogen and sliced with a freezing microtome to obtain rhizosphere soil samples at
distances 0.2, 0.4, 0.6, 0.8, 1.0, 1.5. 2.0, 2.5, 3.5
and 4.5 mm from the mesh surface.
shaken by rotation for 2 h and centrifuged (3000
rpm). Inorganic P in the supernatant was determined immediately by the method of Murphy and
Riley (1962). For determining NaOH-Pi, the residual soil in the centrifuge tube was suspended in 25
ml of 0.1 M NaOH and shaken for 17 h and
centrifuged. Inorganic P in the supernatant was
again determined immediately. Unplanted soil
samples were analyzed as controls.
2.4.1. Root hair determination in soil
To measure root hairs of the cultivars, they
were grown as described above in the rhizosphere
experiment, but six holes (ca. 1 mm) were made at
different places in the nylon screen so that about
six roots penetrated and grew into the soil
columns. After 7 days, the soil columns were
immersed in water in dark at 5°C overnight. The
roots were then removed using a kitchen sieve and
subjected to an ultrasound treatment in an Ultrasound bath (Branson 5200, 120W, 47K Hz) for
5 – 10 min to remove the remaining soil particles
without damaging the root hairs. Root hairs were
measured using Quantimet 500 + Image Processing and Analysis System (Leica) at 10× magnification as described in Gahoonia and Nielsen
(1997).
2.5.3. Plant analysis
Shoots were dried at 80°C to constant weight,
ground and 0.3 g was digested in H2SO4 –H2O2 –
HNO3 mixture. Phosphorus was determined as
described by Murphy and Riley (1962). Phosphorus uptake in field was calculated from shoot dry
matter and P concentrations.
Statistical analysis was performed with Statistical Analysis System Institute (1989) and Microsoft Excel software as found appropriate.
2.4.2. Phosphorus analysis of rhizosphere soil
Soil P was separated into easily desorbable
plant available (NaHCO3-Pi) and strongly adsorbed P (NaOH-Pi) fractions (Hedley et al.,
1982). For determining NaHCO3-Pi, 5 ml of 0.5
M NaHCO3 (pH 8.5) was added to 0.5 g of
air-dry soil in a centrifuge tube. The tubes were
2.5. Field experiment
2.5.1. Experimental design
A randomized complete block design with two
replicates was used. The plot size was 1.5 × 10 m.
Fertilizer applications were 60 kg N and 60 kg K
ha − 1, applied one week before sowing.
2.5.2. Field sampling of plant material
Aerial parts from 1 m2 per plot were harvested
six times during the growing period, i.e. 24, 36,
52, 66, 84 and 94 days after germination.
3. Results
In the nutrient solution experiment, Marinka
produced nearly two times more root length than
Sonate (Table 2), but did not produce more shoot
dry matter. The concentration of P in shoot of
Marinka was lower than Sonate. The concentration of P in the roots of Marinka was higher
indicating that it retained more P in their roots.
The root hair lengths of cultivars were same
(Table 2).
Of the organic acids released from roots of
Marinka and Sonate, the significant differences
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T.S. Gahoonia et al. / Europ. J. Agronomy 12 (2000) 281–289
between the cultivars were found only in release
of citric acid and acetic acid (Fig. 1). Marinka
released three times more citric acid and nearly
Table 2
Root parameters and concentration of phosphorus (P) in
shoot dry matter of two barley cultivars in nutrient solution
experiment
Parameter
Marinka
Sonate
Root length (m g−1)
Root hair length (mm)
Root dry weight (g plant−1)
P in roots (mg g−1)
Shoot dry matter (g plant)
P in shoot dry matter (mg g−1)
109910
0.6790.17
0.529 0.06
3.9 90.3
0.7890.10
3.49 0.4
619 7
0.79 90.20
0.38 90.07
3.0 90.4
0.70 90.04
4.99 0.6
Fig. 3. Time-course of phosphorus uptake (A) and the corresponding shoot dry matter production (B) of two winter barley
cultivars, Marinka and Sonate, in a low-P field.
Fig. 1. Root-released organic acids of two winter barley cultivars, Marinka and Sonate, in sterile nutrient solution culture.
Fig. 2. Depletion of available P (NaHCO3-Pi) and strongly
adsorbed P (NaOH-Pi) in the rhizosphere soil of two winter
barley cultivars in 14 days.
twice as much acetic acid from the roots as compared to Sonate. Both cultivars released large
quantities of fumaric acid (Fig. 1), but the difference between the cultivars was not significant
(P\0.05).
The rhizosphere depletion profiles of easily desorbable plant available P (NaHCO3-Pi) did not
differ, showing that the two cultivars did not
differ in absorbing P from this P fraction (Fig. 2).
However, Marinka decreased the concentration of
strongly adsorbed soil P (NaOH-Pi) by 0.7 mmole
P kg − 1 (from 2.4 to 1.7 mmoles P kg − 1) in the
rhizosphere soil. The decrease was only 0.3 mmole
P kg − 1 soil with Sonate. Marinka could mobilize
and deplete twice as much P from NaOH-Pi than
Sonate (Fig. 2), showing that Marinka possesses
greater ability to release and absorb P from the
strongly adsorbed P in soil.
Under field conditions, over the growth period
of up to 94 days after germination, Marinka
absorbed more P than Sonate (Fig. 3A) and also
produced more shoot dry matter (Fig. 3B).
T.S. Gahoonia et al. / Europ. J. Agronomy 12 (2000) 281–289
4. Discussion
Marinka was superior to Sonate in depletion of
P from strongly adsorbed soil P fraction in the
rhizosphere (Fig. 2) and the P uptake in the field
(Fig. 3A), but not in nutrient solution experiment
(Table 2). Variation in P uptake may result from
variation in root development and/or due to the
ability of the roots to release organic acids into
the rhizosphere. In nutrient solution experiment,
Marinka produced nearly twice as much roots as
Sonate (Table 2). But despite the greater root
length, Marinka neither produced more shoot dry
matter, nor it has a higher plant P concentration
(Table 2). Marinka retained more P in roots.
Hence, total P uptake did not differ between the
cultivars in the nutrient solution experiment,
where all P was readily available in solution without diffusion limitation. The superior P uptake of
Marinka was associated with its ability to extract
P from NaOH-Pi in soil (Fig. 2), as revealed by
rhizosphere studies.
The release of organic acids differed between
the two cultivars (Fig. 1), which corresponded to
the higher depletion of P from the NaOH-Pi (Fig.
2). Citric acid is known to be effective in desorbing strongly bound soil P by ligand exchange
of phosphate against citrate (Gerke, 1992),
whereas acetic acid has only a negligible effect
(Earl et al., 1979). The concentration of soil solution P in the vicinity of roots may reach very low
and close to the Cmin i.e. minimum effective concentration below which nutrient uptake stops
(Nielsen, 1976). When P supply is lowered, enhanced root exudation of organic acids is observed (Lipton et al., 1987). The increased
exudation of organic acids in the vicinity of roots
may release P from the strongly adsorbed P fractions for maintaining soil solution P concentration above the Cmin value (Nielsen and Schjørring,
1983).
The longer root hairs of other barley cultivars
were related to the higher depletion of NaHCO3Pi from rhizosphere soil (Gahoonia and Nielsen,
1997). In contrast, root hair lengths of Marinka
and Sonate were same (Table 2) and the rhizosphere P depletion profiles of NaHCO3-Pi were
not different (Fig. 2). The rhizosphere technique
287
(Gahoonia and Nielsen, 1991) applied to study
depletion of soil P fractions implies that rhizosphere pH remains unchanged (Gahoonia and
Nielsen, 1992). Therefore, the differential ability
of the cultivars to deplete P from NaOH-Pi fractions (Fig. 2) can not be attributed to the root-induced pH change. It may be mentioned that both
NaHCO3 and NaOH, used for fractionation of
soil P, are alkaline, which favor P desorption.
However, the results of this study show that the
higher release of citric acid from roots of Marinka
favors desorption and uptake of P from strongly
adsorbed soil P fraction.
Rhizosphere phosphatase activity may increase
mobilization and depletion of soil organic P (Asmar et al., 1995). Marinka is reported to have
higher activity of root-released extracellular phosphomonoesterase and phosphodiersterase than
Sonate in both sterile and non-sterile conditions
(Asmar and Gissel-Nielsen, 1996). Therefore, the
possibility of higher mobilization of P from soil
organic P by Marinka can not be ruled out, which
might have contributed to the higher P uptake of
Marinka in the field (Fig. 3A). Soils may differ in
available, insoluble and organic P fractions, which
can be more effectively utilized by selecting cultivars with longer root hairs (Gahoonia et al.,
1999), releasing of higher quantity of organic
acids (Fig. 1) and inducing higher phosphatase
activity (Asmar et al., 1995) in the rhizosphere,
respectively.
5. Conclusions
The results showed that the barley cultivar
Marinka releasing more citric acid could extract
and absorb more P from strongly adsorbed soil P
fraction. In tropical regions, where P fertilization
is necessary, a large amount of P is fixed as
strongly adsorbed P in soils. In developed countries, where it is desirable to reduce fertilizer P
inputs, the proportion of P fixed in non-available
P fractions may increase. The barley cultivars
releasing higher amounts of organic acids, such as
citric acid, in the rhizosphere, can have added
advantage of bypassing P fixation process in soils
for utilizing soil and fertilized P more efficiently.
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T.S. Gahoonia et al. / Europ. J. Agronomy 12 (2000) 281–289
This can increase the effectiveness of P fertilization, hence may add to the incentives of using P
fertilizers, where they are necessary. In addition to
the possibility of maintaining yield in low P soils
and the identified P efficient genotypes will also
increase plant gene pools for upgrading the P
efficiency of barley by breeding.
Acknowledgements
The financial support of the Danish Ministry of
Food, Agriculture and Fisheries, under the Research Program ‘Development of Future Crop
Plants’ is thankfully acknowledged.
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